1. Field of the Invention
The present invention relates to a thermostable Flap endonuclease effective for genetic recombination and genetic shuffling based on low homology, as well as to a gene thereof.
2. Description of the Prior Art
There are a method for random mutation of a broad region in a gene and a method for random mutation of a local region in a gene. In the former method, polymerase chain reaction (PCR) is applied such that a specific nucleotide is deleted to induce mutation at the time of replication of the target gene, and in the latter method, PCR using mixed primers is applied to mutate the target site. However, there is still no method for highly efficient induction of genetic recombination or genetic shuffling based on low homology in vitro. A speculative mechanism of genetic recombination and genetic shuffling in vivo is shown in FIG. 1. Step 1 of FIG. 1 shows formation of a single-stranded overhang by a 3'-5' exonuclease; step 2 shows formation of temporary nucleotide base pairs based on low homology; step 3 shows repair of the gap by DNA polymerase and formation of a Flap structure; step 4 shows removal of the Flap single strand by a Flap endonuclease; and step 5 shows ligation of nicks by a DNA ligase. However, the properties of the enzymes catalyzing the respective steps are not fully elucidated. The Flap endonuclease is an enzyme catalyzing step 4. As shown in FIG. 2, the Flap endonuclease specifically recognizes the Flap structure in DNA and cleaves the single strand (called Flap), and this enzyme is found in mammalian cells and yeast cells. Since its origin is organisms living at normal temperatures, this enzyme has poor thermostability and thus is not suitable for artificial genetic shuffling reaction, including PCR.
Because the conventional Flap endonuclease is unstable at high temperatures, it cannot be used to develop methods where genetic recombination or genetic shuffling based on low homology in vitro is induced at high temperatures. However, if a thermostable Flap endonuclease functioning stably at high temperatures can be obtained, it becomes possible to develop new techniques of conducting artificial homologous recombination or genetic shuffling highly efficiently by coupling the enzyme reaction with PCR. Accordingly, development of the thermostable Flap endonuclease stable at high temperature has long been desired.